Long-term goals of this project are to determine the biotin requirements for normal individuals and for individuals in circumstances in which biotin status may be impaired and to investigate the consequences and pathogenic mechanisms of marginal biotin deficiency. Mountingevidence indicates that marginal biotin deficiency is common during normal human gestation and that marginal deficiency is quite teratogenic in some mammalian species. Thus, the following four specific aims are relevant and timely. Specific Aim #1: In our mouse model of teratogenesis we will test the hypothesis that maternal biotin deficiency causes abnormal development of fetal skeleton and palate by causing alterations of fetal gene expression and deficient fetal activities of the biotin-dependentcarboxylases. In biotin deficient and sufficient fetuses, gene expression at several stages of gestation will be screened with microarrays. Affected genes will be confirmed by quantitative real time RT-PCR. Specific Aim #2: In infants with cleft palate or limb shortening, we will test the hypothesis that affected infants have significantly reduced biotin status compared to normal infants. In this case-controlled study, biotin status will be assessed in cord blood using odd-chain fatty acid composition in red blood cell membranes and plasma and by lymphocyte activity of the biotin-dependent enzyme propionyl-CoA carboxylase. Specific Aim #3: In lymphoblast cell culture,we will identify cell surface biotin binding proteins using photoactivatable biotin and affinity chromatography. Candidate putative biotin transporter proteins will be cloned and transfected to confirm function. In studies of lymphoblasts from an individual with biotin transporter deficiency (a new genetic cause of biotin dependency recently discovered by our group), we will investigate the molecular nature of the genetic defect by comparison to normal followed by sequencing of the putative defective transporter. Specific Aim #4: In healthy adult volunteers, we will experimentallyinduce marginal biotin deficiencyusing a modified outpatient design and assess the utility of promising new indicators of biotin status.